The formation of occluding junctions between epithelial cells is essential for the development of a selective transporting epithelial interface between lumenal (apical) and serosal (basolateral) spaces of tissues. The major long-term objectives of this research are to understand the assembly of occluding junctions at a molecular level and to study how their formation is related to the division of the epithelial cell plasma membrane into the biochemically distinct apical and basolateral domains. A functional assay based on the recovery of transepithelial electrical resistance, has been used to identify a monoclonal antibody that inhibits the assembly of occluding junctions between cultured MDCK cells. This antibody recognizes a known cell adhesion molecule, uvomorulin (L-CAM). The major goals of the proposed studies are: 1) To test the hypothesis that uvomorulin dependent intercellular junctions between cultured MDCK cells are required for the formation of the occluding barrier and for the division of the plasma membrane into apical and basolateral domains. 2) To continue using the resistance recovery assay to identify other functional cell surface proteins of occluding junctions, especially protein(s) of the tight junction. 3) To study the role of uvomorulin in the normal in vivo assembly of occluding junctions during the early embryonic development of the toad, Xenopus laevis, with special regards to the formation of the blastocoel cavity. The overall experimental approach is to use functional assays for occluding junction formation in combination with immunological techniques. Proteins and membrane fractions to be used for immunological techniques. Proteins and membrane fractions to be used for immunization will be purified with standard biochemical methods and the distributions of uvomorulin and other proteins will be examined by immunofluorescence microscopy. These studies should improve our understanding of how a selective, polarized epithelial barrier is established and regulated during the normal physiology and development, and how it may be disturbed during pathological processes.